C H A P T E R

14
Innovative Microalgae Pigments as
Functional Ingredients in Nutrition
Efterpi Christaki, Eleftherios Bonos, Panagiota Florou-Paneri
Laboratory of Nutrition, School of Veterinary Medicine, Faculty of Health Sciences,
Aristotle University of Thessaloniki, Thessaloniki, Greece

1. INTRODUCTION in biotechnology (Gouveia et al., 2008; Christaki

et al., 2011; Borowitzka, 2013).
Isolating and identifying bioactive com-
pounds from marine microalgae have emerged
as a recent trend, mainly to develop new func- 2. MICROALGAE
tional foods. The interest in functional food in-
gredients derived from microalgae in relation For centuries, microalgae have been recog-
to nutritional genomics (nutrigenomics and nized as the basis of the food chain in aquatic
nutrigenetics) aims to minimize the risk of ecosystems. They are primitive prokaryotic
chronic diseases that have a genetic predisposi- (e.g., cyanobacteria: blue-green algae) or eukary-
tion (Simopoulos, 2010; Cadoret et al., 2012; otic (e.g., chlorophyta: green algae) photosyn-
Freitas et al., 2012). From a biotechnological thetic microorganisms, which range in diameter
point of view, a great deal of interest has from 0.2 to 2.0 mm (Richmond, 2004; Lordan
been expressed for the commercial large-scale et al., 2011). Because of their simple structure,
production of microalgae; several different they can grow rapidly and live in harsh environ-
research fields are exploring the use of algal mental conditions such as heat, cold, anaerobi-
biomass. osis, salinity, and exposure to ultraviolet
Due to their abundant availability in the radiation (Richmond, 2004; Batista et al., 2013).
aquatic ecosystem, microalgae have the potential Microalgae, as an alternative to higher plants,
to become excellent sources of high-biological- have the ability to be cultivated on nonarable
value compounds with health benefits, such as land or seashores; consequently, they do not
lipids, polyunsaturated fatty acids, proteins, vi- compete with food and feed cultivation. For
tamins, minerals, and pigments, which can be these reasons, microalgal biomass is attracting
used in different markets. Thus, microalgae worldwide attention to satisfy the so-called
represent an important and dynamic new area bioeconomy demand (Gouveia et al., 2008;

Handbook of Marine Microalgae

http://dx.doi.org/10.1016/B978-0-12-800776-1.00014-5 233 © 2015 Elsevier Inc. All rights reserved.
234 14. INNOVATIVE MICROALGAE PIGMENTS AS FUNCTIONAL INGREDIENTS IN NUTRITION

Christaki et al., 2013). Some edible microalgae enzyme-assisted extraction, microwave-

are already commercialized and being used assisted extraction, ultrasound-assisted extrac-
in biotechnology, including Chlorophyta tion, supercritical fluid extraction, and
(green algae), Rhodophyta (red algae), and Cya- pressurized liquid extraction (Plaza et al.,
nobacteria (blue-green algae) (Spolaore et al., 2009; Kadam et al., 2013; Reyes et al., 2014).
2006; Christaki et al., 2010, 2011; Mulders et al., Therefore, to make microalgal biomass econom-
2014). ically feasible, it is mandatory to develop
The cultivation of microalgae can be per- the technology and to optimize the use of
formed in open-culture systems (e.g., lakes, la- all produced substances (Vanthoor-Koopmans
goons, artificial ponds) and highly controlled and Wijffels, 2013; Wijffels et al., 2013).
closed-culture systems (photobioreactors) (Mata
et al., 2010; Wijffels et al., 2013). Closed systems
offer better options for the growth of most micro- 3. PIGMENTS FROM MICROALGAE
algae strains, providing the ability to protect the
culture from contamination by undesirable One of the most prominent characteristics of
microorganisms, control processing conditions, algae is their color, which is determined by their
and allow higher productivity and better quality pigments. These pigments are colorful chemical
for the biomass; however, they are expensive. substances that are part of the photosynthetic
Consequently, much effort has been expended system of microalgae. They are distinguished
toward finding more cost-efficient modes of into three classes: carotenoids (0.1e0.2% of dry
microalgal mass cultivation with regard to weight on average, and up to 14% of dry weight
open systems. The main advantage of open sys- for b-carotene in some species), chlorophylls
tems is their use of sunlight to produce biomass (0.5e1.0% of dry weight), and phycobiliproteins
and lower costs to build and operate. On the (up to 8% of dry weight) (Gouveia et al., 2008; de
other hand, environmental factors are not fully Jesus Raposo et al., 2013; Markou and Nerantzis,
controlled (Freitas et al., 2012; Barra et al., 2013; Barra et al., 2014).
2014). Recently, some new concepts for new To be deemed suitable for producing pig-
bioreactors have been proposed (e.g., hybrid ments commercially, microalgae strains have to
systems combining open ponds and photobior- meet various criteria, such as ease of culture,
eactors, floating photobioreactors); most of lack of toxicity, high nutritional value, and pres-
them aim to reduce costs (Norsker et al., 2011; ence of digestible cell walls to make the nutrients
Zittelli et al., 2013). available. As shown in Table 1, the most
Microalgae could be used as whole frequently used species are Dunaliella salina, Hae-
biomass without extraction because their matococcus pluvialis, Chlorella spp., Muriellopsis
bioactive compounds are more stable in their spp., Scenedesmus spp., Spirulina (Arthrospira)
natural matrix than in the extracts (Gouveia spp., and Porphyridium spp. (Eonseon et al.,
and Empis, 2003; Christaki et al., 2013). Tradi- 2003; Herrero et al., 2006; Spolaore et al., 2006;
tionally, microalgae pigments are extracted Hu et al., 2008; Mata et al., 2010; Borowitzka,
with solvents after breaking the cell walls 2013).
through mechanical or thermal treatments
(Freitas et al., 2012; Batista et al., 2013). In recent
3.1 Carotenoids
years, in order to obtain bioactive compounds
from microalgae, some novel environmentally Probably the most commercially valuable
friendly and efficient selective extraction tech- groups of algae pigments are the carotenoids.
nologies have been developed, such as They are isoprenoid molecules, yellow to red in
 3. PIGMENTS FROM MICROALGAE 235
TABLE 1 Commercially Interesting Pigments Across Another distinction of carotenoids can be
the Most Common Microalgae Groups made between primary and secondary caroten-
Microalgae Pigments References
oids. Primary ones, such as b-carotene and
lutein, are structural and functional components
Dunaliela salina b-carotene, Plaza et al. (2009); of the photosynthetic apparatus and are directly
zeaxanthin, Herrero et al. (2006);
involved in photosynthesis. They have a light-
chlorophylls a, b Hu et al. (2008)
harvesting role as well as a photoprotective
Haematococcus astaxanthin, Plaza et al. (2009); one, which can be expressed either by filtering,
pluvialis canthaxanthin, Herrero et al. (2006);
quenching, or scavenging (Christaki et al.,
lutein, Jaime et al. (2010);
chlorophylls a, b Batista et al. (2013) 2013; Skjanes et al., 2013; Latowski et al., 2014;
Mulders et al., 2014).
Chlorella spp. astaxanthin, Wu et al. (2007);
Secondary carotenoids, including astaxanthin
canthaxanthin, Mulders et al. (2014)
chlorophylls a, b and canthaxanthin, are not bound to the photo-
synthetic apparatus. They are synthesized in
Scenedesmus lutein, Del Campo et al.
the chloroplast and accumulate in the cytoplasm
spp. b-carotene (2007); Plaza et al.
(2009) (Eonseon et al., 2003; Mulders et al., 2014). Some
environmental and cultural factors, such as high
Muriellopsis lutein Del Campo et al.
light density, nutrient starvation, temperature
spp. (2007); Fernandez-
Sevilla et al. (2010) changes, pH values, high salt concentration,
and oxidative stress, exclusively induce the over-
Spirulina spp. b-carotene, Maoka (2011);
production of secondary carotenoids as an adap-
zeaxanthin, Eriksen (2008); de
phycocyanin, Jesus Raposo et al. tation to the above conditions. Thus, these
allophycocyanin (2013) carotenoids, produced in microalgae via carote-
nogenesis process, act only as photoprotective
Porphyridium phycoerythrin Borowirka (2013);
spp. Rodriguez-Sanchez pigments with a filtering role so they can defend
et al. (2012); Plaza themselves (e.g., from harmful radiation;
et al. (2009) Lemoine and Schoefs, 2010; Takashi, 2011;
Christaki et al., 2013; Skjanes et al., 2013;
Mulders et al., 2014). Chlorophyta microalgae
color, and were some of the first pigments in na- is the only group known to contain species that
ture. Because the carotenoids are lipid-soluble overproduce secondary carotenoids (Lamers
substances, they are absorbed with fats and enter et al., 2008; Mulders et al., 2014). Nevertheless,
the circulation in the blood bound to different li- there is a lack of data about metabolic pathways
poproteins (Lordan et al., 2011; Christaki et al., in microalgae for the overproduction of both pri-
2013). Humans and animals (with the exception mary and secondary carotenoids, because the
of an aphid genus) are incapable of synthesizing genes and enzymes involved are largely unpre-
carotenoids and hence require them in their diet dictable (Takashi, 2011; Mulders et al., 2014).
(Moran and Jarvik, 2010; Christaki et al., 2013; b-Carotene was the first high-value product
Latowski et al., 2014). Chemically, carotenoids commercially produced from the green algae
can be divided into two groups: the carotenes Dunaliella salina, which is its richest source
(hydrocarbons; e.g., a-carotene, b-carotene, lyco- (Herrero et al., 2006; Gouveia et al., 2008; Hu
pene) and the xanthophylls (oxygenated mole- et al., 2008; Borowitzka, 2013; Dewapriya and
cules; e.g., astaxanthin, lutein, canthaxanthin; Kim, 2014). Natural b-carotene is a mixture of
Eonseon et al., 2003; Guedes et al., 2011; all-trans and 9-cis isomers, which is rarely ob-
Christaki et al., 2013). tained in synthetic carotenoids. Although it is
236 14. INNOVATIVE MICROALGAE PIGMENTS AS FUNCTIONAL INGREDIENTS IN NUTRITION

in competition with the less-expensive synthetic Lutein is a yellow primary carotenoid found
form, it is often preferred because it is considered in microalgae species, such as Scenedesmus
to have anticancer activity (Gouveia et al., 2008; (4.5e5.5 mg/g) and Chlorella (4.6 mg/g) (Plaza
Lordan et al., 2011; Borowitzka, 2013). Further- et al., 2009). Another microalgae species, Muriel-
more, natural b-carotene produced by algae is lopsis spp., was found to possess high lutein con-
absorbed better by the body compared to the tent (up to 35 mg/l) that does not seem to be
synthetic form (Lordan et al., 2011; Skjanes triggered under stress conditions, coupled with
et al., 2013). a high growth rate. Therefore, this microalga
Although b-carotene is a primary carotenoid, could be exploited for commercial production
under stress conditions, it can act as a secondary of this carotenoid (Fernandez-Sevilla et al.,
carotenoid. For example, in the cultivation of 2010; Guedes et al., 2011; Skjanes et al., 2013).
Dunaliela salina under high salinity, light stress, Lutein, apart from being a strong antioxidant,
and nitrogen starvation, b-carotene is accumu- is used as a natural colorant in the feed industry,
lated to more than 14% of the microalgae dry in drugs, and in cosmetics (Wu et al., 2007; Plaza
weight, the highest content in the known sources et al., 2009; de Jesus Raposo et al., 2013).
(Del Campo et al., 2007; Lamers et al., 2012). Canthaxanthin, a secondary carotenoid, is
Other researchers (Gomez and Gonzalez, 2005; produced in large quantities by Chlorella spp.
Coesel et al., 2008) reported that the shift in low and Scenedesmus spp. under salt stress,
culture temperature of Dunaliela from 30  C to nitrogen deprivation, and ultraviolet irradiation
10  C resulted in a twofold increase of b-carotene (Takaichi, 2011; Skjanes et al., 2013).
and 9-cis-b-carotene, while increasing the salt
concentration from 4% to 9% had a 30-fold in-
3.2 Chlorophylls
crease in the accumulation of b-carotene.
Astaxanthin derived from microalgae is gain- Chlorophyll, present in all higher plants, is a
ing importance commercially. It can be pro- valuable bioactive constituent that can be
duced under stress conditions from the green extracted from the microalgal biomass. It is a
freshwater alga Haematococcus pluvialis, which green pigment that is ubiquitous in nature
contains up to 0.2e3.0% on a dry weight basis; because it is responsible for the photosynthetic
this is currently the prime natural source of process (Hosikian et al., 2010; Stengel et al.,
astaxanthin (Dufosse, 2009; Jaime et al., 2010; 2011). Nowadays, there is a strong focus on the
Chandi and Gill, 2011; Batista et al., 2013). This commercial production of dhlorophyll as a natu-
red-orange secondary xanthophyll is a powerful ral pigment in the food and feed industries, in
biological antioxidant that occurs in nature, pro- pharmaceuticals, and in cosmetics (Hosikian
tecting membranous phospholipids and other et al., 2010; Stengel et al., 2011). Chlorophyll is
lipids against peroxidation (Hussein et al., a tetrapyrrole with a centrally bound magne-
2006; Mata et al., 2010). Furthermore, astaxan- sium ion (Pangestuti and Kim, 2011; Mulders
thin has some additional advantages over other et al., 2014).
carotenoids: it is more stable, it can easily cross There are several kinds of chlorophylls in
the bloodebrain barrier, and it has high tincto- microalgaedchlorophylls a, b, c, d and fdwhich
rial properties (Dufosse, 2009). Astaxanthin is have some small differences in their absorption
probably best known for eliciting the pinkish- spectra and consequently their tonality. Chloro-
red color in the flesh of salmonoids, shrimp, lob- phyll a has a blue-green color, chlorophyll b is
sters, and crayfish (Eonseon et al., 2003; Chu, a brilliant green, chlorophyll c is yellow-green,
2012), representing 74e98% of their total pig- chlorophyll d is a brilliant/forest green, and
ments (Lordan et al., 2011). chlorophyll f is emerald green (Chen et al.,
 4. BIOLOGICAL ACTIVITIES OF MICROALGAE PIGMENTS AND HEALTH BENEFIT EFFECTS 237
2010; Roy et al., 2011). Chlorophyll a, which is which differ in their spectral properties (Wright
the major light-harvesting pigment, appears in and Jeffrey, 2006; Eriksen, 2008; Blot et al.,
all photosynthetic organisms. Chlorophyll b ap- 2009; Chu, 2012; Freitas et al., 2012; Mulders
pears exclusively in chlorophyta and their et al., 2014). In addition to being natural food col-
descendants, whereas chlorophyll c appears orants, these phycobiliproteins are strongly fluo-
exclusively in rhodophyta (Jeffrey and Wright, rescent markers and have antioxidant properties
2005). (Eriksen, 2008; Stengel et al., 2011; de Jesus
Although chlorophyll is a natural pigment, Raposo et al., 2013). Also, the above-mentioned
there are some disadvantages associated with substances can neutralize the reactive oxygen
its use. For example, it is unstable in foods where species (ROS) due to their chemical structures
it is incorporated under different pH conditions and chelating properties, thus reducing oxida-
(Hosikian et al., 2010). Additionally, when the tive stress (Roy et al., 2007; Rodriguez-Sanchez
magnesium ion is lost, chlorophyll becomes et al., 2012).
pale and dusky colored (Humphrey, 2004). Phycocyanin from Spirulina spp. and phycoer-
It has been reported that the chlorophylls may ythrin from Porphyridium spp. are two of the
not need to be overproduced to make a most well-known phycobiliproteins that have
microalgal-based production process economi- been produced commercially (Plaza et al., 2009;
cally feasible (Mulders et al., 2014). Conse- Rodriguez-Sanchez et al., 2012; Borowitzka,
quently, rapidly growing species like Chlorella 2013).
can contain the two main types of chlorophylls The content of these protein-bound unique
(a, b), up to 4.5% of dry weight, and therefore pigments found in microalgae is degraded under
could be the most attractive production some stress conditions, such as phosphorus, ni-
material when grown under optimal conditions trogen, and sulfur starvation (Eriksen, 2008;
(Cuaresma et al., 2011; Miazek and Ledakowicz, Hifney et al., 2013). Nevertheless, the increase
2013). On the other hand, it has been reported in salt concentration up to 0.6 M resulted in an
that chlorophyll content in microalgal biomass elevation of the total phycobiliprotein content
is reduced significantly under stress conditions in Spirulina spp., from 25% to 45% of dry matter,
(Markou and Nerantzis, 2013). while a further increase to 0.9 M salt affected
negatively the phycobiliprotein synthesis
(Hifney et al., 2013).
3.3 Phycobiliproteins
Phycobiliproteins are deep-colored, water-
soluble proteins that are present mainly in cya- 4. BIOLOGICAL ACTIVITIES OF
nobacteria and rhodophyta. They capture light MICROALGAE PIGMENTS AND
energy, which is then passed on to chlorophylls HEALTH BENEFIT EFFECTS
during photosynthesis. Phycobiliproteins are
composed of proteins and covalently bound via Several studies have focused on the use of nat-
cysteine amino acid chromophores called phyco- ural pigments derived from microalgae, because
bilins, belonging to open-chain tetrapyrroles they have health-promoting properties and a
(Eriksen, 2008; de Jesus Raposo et al., 2013; broad range of potential industrial applications.
Watanabe and Ikeuchi, 2013; Mulders et al., Consumers are becoming increasingly aware of
2014). Phycobiliproteins that are not essential the correlation between diet, health, and disease
for the function of cells include phycocyanin prevention; thus, microalgae represent an impor-
(blue pigment), phycoerythrin (red pigment), tant and dynamic new area in biotechnology
and allophycocyanin (light-blue pigment), (Figure 1).
238 14. INNOVATIVE MICROALGAE PIGMENTS AS FUNCTIONAL INGREDIENTS IN NUTRITION

FIGURE 1 Distribution, biological activities, and commercial applications of natural pigments derived from microalgae.

Because humans and animals cannot synthe- lipophilic part from lipid peroxidation or scav-
size pigments de novo, these are either provided enging ROS in photo-oxidative processes (Stahl
by the diet or partly modified through metabolic and Sies, 2003; Christaki et al., 2011; Lordan
reactions from precursor substances. For et al., 2011; Pangestuti and Kim, 2011). However,
example, b-carotene can be metabolized to it has been reported that b-carotene might act as
vitamin A (retinol). b-Carotene has a very high a pro-oxidant in the process of lipid peroxidation
pro-vitamin A activity because every molecule when there is a high oxygen pressure and high
of this compound produces two molecules of carotenoid amount (Polyakov et al., 2001; Stahl
retinol (Liaanen-Jensen, 1998; Graham and and Sies, 2003).
Rosser, 2000; Christaki et al., 2013). Apart from their high concentration in the
Regarding b-carotene and astaxanthin, and to microalgal biomass and the even larger quanti-
a lesser degree other microalgae pigments, it ties produced by controlling some environ-
must be noticed that they have strong antioxi- mental conditions, the antioxidants from
dant activity, which aims to mediate the harmful microalgae can act synergistically to increase
effects of free radicals by protecting the the positive effects on human health (Stahl and
 5. NEW TRENDS IN COMMERCIAL APPLICATIONS OF MICROALGAE PIGMENTS 239
Sies, 2003; Heydarizadeh et al., 2013). These pig- Studies both in vitro and in vivo have shown
ments have greater antioxidant effects than the cancer-preventive effects of chlorophylls,
vitamin E, but weaker than synthetic commercial with particular emphasis on their antimutagenic
antioxidants, such as butylated hydroxytoluene effects (Ferruzi and Blakeslee, 2007; Gouveia
or butylated hydroxyanisole (Natrah et al., et al., 2008; Hosikian et al., 2010). The protective
2007; Pangestuti and Kim, 2011). The use of syn- effects of carotenoids and phycobiliproteins on
thetic antioxidants is under strict regulation, the development of cancerous tumors and leuke-
especially in the European Union countries, mia have also been reported (Plaza et al., 2009;
due to their potential health hazards. Therefore, Vilchez et al., 2011; Heydarizadeh et al., 2013).
natural antioxidants can be used as safe alterna- Microalgae pigments are able to boost the im-
tives in the industry (Spolaore et al., 2006; Gou- mune system (e.g., antibody production) and
veia et al., 2008). to act as anti-inflammatory agents against
Dietary intake of marine microalgae-derived asthma, ulcers, arthritis, and muscle damage
antioxidants has shown the ability to protect or- (by providing increased muscle endurance;
ganisms against various chronic disorders, such Mata et al., 2010; Guedes et al., 2011; Stengel
as cancer, diabetes, atherosclerosis, coronary dis- et al., 2011; de Jesus Raposo et al., 2013), as
ease, ischemic brain development, metabolic well as antiproliferative agents (Plaza et al.,
syndromes, gastrointestinal and liver diseases, 2009; Lordan et al., 2011). In addition, some
as well as neurodegenerative diseases such as epidemiological data explain the above proper-
Alzheimer disease and Parkinson disease ties of carotenoids, which could directly act to-
(Riccioni et al., 2011; Cadoret et al., 2012; ward the DNA to regulate the production of
Gouveia, 2014; Martins et al., 2014), or to amelio- RNA (Guerin et al., 2003; Hussein et al., 2006).
rate cognitive functions (Kidd, 2011). According Moreover, chlorophylls seem to be a good
to Guerin et al. (2003), astaxanthin could be approach for the treatment of ulcers and postop-
effective against human prostatic hyperplasia erative wounds (Hosikian et al., 2010). Also, pa-
and prostatic cancer through the enzyme tients with pancreatitis can possibly be treated
5-a-reductase, which is involved in the abnormal with chlorophyll a (Yaakob et al., 2014).
prostate growth. Other researchers have
described the use of phycocyanin as a nephro-
protector (Rodriguez-Sanchez et al., 2012) or a 5. NEW TRENDS IN COMMERCIAL
protector of human pancreatic cells (Chu, 2012). APPLICATIONS OF MICROALGAE
In addition, the carotenoids lutein and zeax- PIGMENTS
anthin, due to their antioxidant capacity, can
protect the eye macula from adverse photochem- Pigments from microalgae are now strongly
ical reactions (Friedman et al., 2004), while b- demanded by the market as renewable natural
carotene helps to prevent premature aging color enhancers for foods and feeds, which
caused by ultraviolet radiation (Miyashita, simultaneously provide certain health benefits.
2009; Fernandez-Sevilla et al., 2010) or to treat Furthermore, these pigments, which are particu-
skin melasma (Yaakob et al., 2014). Generally, larly strong dyes even at very low levels (parts
b-carotene, apart from limiting photooxidative per million), have important applications in the
damage to the skin, provides protection against pharmaceutical industry (e.g., as fluorescence-
sunburns (erythema solare) (Eonseon et al., based indicators, as biochemical tracers in im-
2003; Stahl and Sies, 2003). Moreover, in marine mune assays) and in the cosmetic industry
animals, b-carotene is stored in the gonads, so it (e.g., as skin cream to stimulate collagen
is essential for reproduction (Maoka, 2011). synthesis).
240 14. INNOVATIVE MICROALGAE PIGMENTS AS FUNCTIONAL INGREDIENTS IN NUTRITION

The development of foods with attractive ap- example, astaxanthin is recognized as a food
pearances is an important goal in the food and colorant by the Food and Drug Administration
feed industries. b-Carotene produced by micro- in the United States, in Japan, and in some
algae serves as one of the most utilized food col- European countries, it but has not yet been
oring agents in pasta, fruit juices, soft drinks, approved in the European Union countries
confectionary, margarine, dairy products, and (Lordan et al., 2011; Borowitzka, 2013; Ambati
salad dressings (Gouveia et al., 2008; Plaza et al., 2014). Pigments derived from microalgae
et al., 2009; Christaki et al., 2011; Guedes et al., are unique, so extensive assessment tests (e.g.,
2011; Christaki et al., 2013). Phycocyanin, with acute toxicity, mutagenicity, teratogenicity,
its blue color, can be used in various food prod- embryotoxicity, and reproductive toxicity) to
ucts such as chewing gums, candies, dairy prod- confirm their safety for consumers need to be
ucts, jellies, ice creams, and beverages; its color is considered before their introduction into the
stable in dry preparations, but it is sensitive to market.
high temperatures and light (Gouveia et al., Apart from the conventional (nontransgenic)
2008; Dufosse, 2009). Phycoerythrin, with its approach, the biotechnological production of pig-
red color, can be used for the pigmentation of ments and other valuable substances from micro-
confections, gelative desserts, and dairy prod- algae with the use of genetic engineering is a very
ucts. This color is stable when phycoerythrine tempting alternative. Such an approach may result
is incorporated in dry food preparations (stored in improvements of nutritional value and optimi-
under low humidity) and has a long shelf life at zation of metabolite production with functional
pH 6e7 (Dufosse et al., 2005). In addition, properties, which makes these substances better
Chandi and Gill (2011) reported that novel suited for commercial applications (Freitas et al.,
food applications of pigments can include to- 2012; Htet et al., 2013; Rasala et al., 2014). Never-
mato ketchup, processed meats such as sausage theless, the use of transgenic microalgae faces
and ham, and marine products such as fish paste some problems and challenges, at least in Europe,
and surimi. It is important to notice that microal- such as competitiveness, public acceptance, regu-
gae pigments in foods besides the coloring and latory issues, and biosafety concerns (Freitas
nutritional purposes can also cause significant et al., 2012; Htet et al., 2013; Rasala et al., 2014).
changes in the rheological properties (Gouveia Generally, genetically modified food is viewed
et al., 2008). with a large degree of skepticism by consumers.
Carotenoids are also used as feed additives in
the commercial rearing of many aquatic organ-
isms to enhance the reddish color of the flesh 6. CONCLUSIONS
of salmon, trout, and shrimp, as well as feed sup-
plement for some types of zooplankton (e.g., ro- Microalgae pigments such as carotenoids,
tifers and copepods (Plaza et al., 2009; Mata chlorophylls, and phycobiliproteins could be a
et al., 2010; Guedes et al., 2011)). Also, microal- leading natural resource for innovative potential
gae pigments could be used to enrich the functional ingredients in nutrition. Nowadays,
yellowish color of egg yolk and chicken skin these natural pigments are preferred over syn-
(Plaza et al., 2009; Mata et al., 2010; Guedes thesized substances and are finding commercial
et al., 2011) or to improve the appearance of applications, mainly in the food and feed indus-
pet foods (Spolaore et al., 2006; Skjanes et al., tries. Nevertheless, some bottlenecks, such as
2013). high production costs and low yields, need to
Laws and regulations concerning food addi- be solved before microalgae can be moved
tives and functional foods vary by country. For from niche markets to large-scale use.
 REFERENCES 241

References de Jesus Raposo, M.F., de Morais, R.M.S.C., de

Morais, A.M.M.B., 2013. Health applications of bioactive
Ambati, R.R., Moi, P.S., Ravi, S., Aswathanarayana, R.G., compounds from marine microalgae. Life Sci. 93,
2014. Astaxanthin: sources, extraction, stability, biological 479e486.
activities and its commercial applicationsda review. Mar. Del Campo, J.A., Garcia-Gonzalez, M., Guerrero, M.G., 2007.
Drugs 12, 128e152. Outdoor cultivation of microalgae for carotenoid produc-
Barra, L., Chandrasekaran, R., Corato, F., Brunet, C., 2014. tion: current state and perspectives. Appl. Microbiol.
The challenge of ecophysiological biodiversity for Biotechnol. 74, 1163e1174.
biotechnological applications of marine microalgae. Dewapriya, D., Kim, S.K., 2014. Marine microorganisms: an
Mar. Drugs 12, 1641e1675. emerging avenue in modern nutraceuticals and functional
Batista, A.P., Gouveia, L., Bandarra, N.M., Franco, J.M., foods. Food Res. Int. 56, 115e125.
Raymundo, A., 2013. Comparison of microalgal biomass Dufosse, L., 2009. Microbial and microalgal carotenoids as col-
profiles as novel functional ingredient for food ourants and supplements. In: Britton, G., Liaanen-
products. Algal Res. 2, 164e173. Jensen, S., Pfander, H. (Eds.), Carotenoids, Nutrition and
Blot, N., Wu, X.J., Thomas, J.C., Zhang, J., Garczarek, I., Health, Volume 5. Birkhauser Verlag, Basel, Switzerland.
Bohm, S., Tu, J.M., Zhou, M., Ploscher, M., Eichacker, L., Dufosse, L., Galaup, P., Yaron, A., Arad, S.M., Blanc, P.,
Partensky, F., Scheer, H., Zhao, K.H., 2009. Phycourobilin Murthy, N.C., Ravishankar, G.A., 2005. Microorganisms
in trichromatic phycocyanin from oceanic cyanobacteria and microalgae as sources of pigments for food use: a sci-
is formed post-translationally by a phycoerythrobilin entific oddity or an industrial reality? Trends Food Sci.
lyase-isomerase. J. Biol. Chem. 284, 9290e9298. Technol. 16, 389e406.
Borowitzka, M.A., 2013. High-value products from Eonseon, J., Polle, J.E.W., Lee, H.K., Hyund, S.M., Chang, M.,
microalgaedtheir development and commercialisation. 2003. Xanthophylls in microalgae: from biosynthesis to
J. Appl. Phycol. 25, 743e756. biotechnological mass production and application.
Cadoret, J.P., Garnier, M., Saint-Jean, B., 2012. Microalgae, J. Microbiol. Biotechnol. 13, 165e174.
functional genomics and biotechnology. Adv. Bot. Res. Eriksen, N.T., 2008. Production of phycocyaninda pigment
64, 285e341. with applications in biology, biotechnology, foods and
Chandi, G.K., Gill, B.S., 2011. Production and characteriza- medicine. Appl. Microbiol. Biotechnol. 80, 1e14.
tion of microbial carotenoids as an alternative to synthetic Fernandez-Sevilla, J.M., Fernandez, A.F.G., Grima, M.E.,
colors: a review. Int. J. Food Prop. 14, 503e513. 2010. Biotechnological production of lutein and its
Chen, M., Schliep, M., Willows, R.D., Cai, Z.L., Neilan, B.A., applications. Appl. Microbiol. Biotechnol. 86, 27e40.
Scheer, H., 2010. A red-shifted chlorophyll. Science 329, Ferruzi, M.G., Blakeslee, J., 2007. Digestion, absorption, and
1318e1319. cancer preventive activity of dietary chlorophyll
Christaki, E., Bonos, E., Giannenas, I., Florou-Paneri, P., 2013. derivatives. Nutr. Res. 27, 1e12.
Functional properties of carotenoids originating from Freitas, A.C., Rodrigues, D., Rocha-Santos, T.A.P.,
algae. J. Sci. Food Agric. 93, 5e11. Gomes, A.M.P., Duarte, A.C., 2012. Marine biotechnology
Christaki, E., Florou-Paneri, P., Bonos, E., 2011. Microalgae: a advances towards applications in new functional foods.
novel ingredient in nutrition. Int. J. Food Sci. Nutr. Biotechnol. Adv. 30, 1506e1515.
62, 794e799. Friedman, D.S., O’Colmain, B.J., Mu~ noz, B., Tomany, S.C.,
Christaki, E., Karatzia, M., Florou-Paneri, P., 2010. The use of McCarty, C., De Jong, P.T., Nemesure, B., Mitchell, P.,
algae in animal nutrition. J. Hellenic Vet. Med. Soc. Kempen, J., Congdon, N., 2004. Prevalence of age-
61, 267e276. related macular degeneration in the United States. Arch.
Chu, W.L., 2012. Biotechnological applications of microalgae. Ophthalmol. 122, 564e572.
Int. E-J. Sci. Med. Educ. 6, S24eS37. Gomez, P., Gonzalez, M., 2005. The effect of temperature
Coesel, S.N., Baumfartner, A.C., Teles, L.M., Ramos, A.A., and irradiance on the growth and carotenogenic capac-
Henriques, N.M., Cancela, L., Varela, J.C.S., 2008. ity of seven strains of Dunaliella salina (Chlorophyta)
Nutrient limitation is the main regulatory factor for cultivated under laboratory conditions. Biol. Res. 38,
carotenoid accumulation and for Psy and Pds steady 151e162.
state transcript levels in Dunaliella salina (Chlorophyta) Gouveia, L., 2014. From tiny microalgae to huge biorefineries.
exposed to high light and salt stress. Mar. Biotechnol. Oceanography 2, 120.
10, 602e611. Gouveia, L., Batista, A.P., Sousa, I., Raymundo, A.,
Cuaresma, M., Janssen, M., Vilchez, C., Wijffels, R.H., 2011. Bandarra, N.M., 2008. Microalgae in novel food
Horizontal or vertical photobioreactors? How to improve products. In: Papadopoulos, K.N. (Ed.), Food Chemistry
microalgae photosynthetic efficiency. Biores. Technol. Research Developments. Nova Science Publishers Inc.,
102, 5129e5137. Hauppauge, NY, pp. 75e112.
242 14. INNOVATIVE MICROALGAE PIGMENTS AS FUNCTIONAL INGREDIENTS IN NUTRITION

Gouveia, L., Empis, J., 2003. Relative stabilities of microalgal Kadam, S.U., Tiwari, B.K., O’Donnell, C.P., 2013. Application
carotenoids in microalgal extracts, biomass and fish feed: of novel extraction technologies for bioactives from
effect of storage conditions. Innov. Food Sci. Emerging marine algae. J. Agric. Food Chem. 61, 4667e4675.
Technol. 4, 227e233. Kidd, P., 2011. Astaxanthin, cell membrane nutrient with
Graham, R.D., Rosser, J.M., 2000. Carotenoids in staple foods: diverse clinical benefits and anti-aging potential. Altern.
their potential to improve human nutrition. Food Nutr. Med. Rev. 16, 355e364.
Bull 21. Lamers, P.P., Janssen, A.M., de Vos, R.G.H., BIno, R.J.,
Guedes, A.C., Amaro, H.M., Malcata, F.X., 2011. Microalgae Wijffels, R.H., 2008. Exploring and exploiting carotenoid
as sources of carotenoids. Mar. Drugs 9, 625e644. accumulation in Dunaliella salina for cell-factory
Guerin, M., Huntley, M.E., Olaizola, M., 2003. Haematococcus applications. Trends Biotechnol. 26.
astaxanthin: applications for human health and nutrition. Lamers, P.P., Janssen, A.M., de Vos, R.G.H., BIno, R.J.,
Trends Biotechnol. 21, 210e216. Wijffels, R.H., 2012. Carotenoid and fatty acid metabolism
Herrero, M., Jaime, L., Martin-Alvarez, P.J., Cifuentes, A., in nitrogen-starved Dunaliella salina, a unicellular green
Ibanez, E., 2006. Optimization of the extraction of antiox- microalga. J. Biotechnol. 162, 21e27.
idant from Dunaliella salina microalga by pressurized Latowski, D., Szymanska, R., Kazimierz, S., 2014. Caroten-
liquids. J. Agric. Food Chem. 54, 5597e5603. oids involved in antioxidant system of chloroplasts. In:
Heydarizadeh, P., Poirier, I., Loizeau, D., Ulmann, L., Ahmad, P. (Ed.), Oxidative Damage to Plants: Antioxi-
imouni, V., Schoefs, B., Bertrand, M., 2013. Plastids of dant Networks and Signaling. Academic Press, Waltham,
marine phytoplankton produce bioactive pigments and MA, pp. 289e319.
lipids. Mar. Drugs 11, 3425e3471. Lemoine, Y., Schoefs, B., 2010. Secondary ketocarotenoid
Hifney, A.F., Issa, A.A., Fawzy, M.A., 2013. Abiotic stress astaxanthin biosynthesis in algae: a multifunctional
induced production of b-carotene, allophycocyanin response to stress. Photosynthesis Res. 106, 155e177.
and total lipids in Spirulina sp. J. Biol. Earth Sci. 3, Liaanen-Jensen, S., 1998. Carotenoids in food chain. In:
B54eB64. Britton, G., Liaanen-Jensen, S., Pfander, H. (Eds.), Carot-
Hosikian, A., Lim, S., Halim, R., Danquah, M.K., 2010. enoids: Biosynthesis and Metabolism, Vol. 3. Birkhauser,
Chlorophyll extraction from microalgae: a review on Basel, Switzerland, pp. 359e371.
the process engineering aspects. Int. J. Chem. Eng. Lordan, S., Paul Ross, R., Stanton, C., 2011. Marine bioac-
2010, 11. tives as functional food ingredients: potential to reduce
Htet, M.Z., Ling, L.Y., Yun, S.H., Rajee, O., 2013. Biofuel from the incidence of chronic diseases. Mar. Drugs 9,
microalgaeda review on the current status and future 1056e1100.
trends. Int. J. Adv. Biotechnol. Res. 4, 329e341. Maoka, T., 2011. Carotenoids in marine animals. Mar. Drugs
Hu, C.C., Lin, J.T., Lu, F.J., Chou, F.P., Yang, D.J., 2008. Deter- 9, 278e293.
mination of carotenoids in Dunaliella salina cultivated in Markou, G., Nerantzis, E., 2013. Microalgae for high-value
Taiwan and antioxidant capacity of the algal carotenoid compounds and biofuels production: a review with focus
extract. Food Chem. 109, 439e446. on cultivation under stress conditions. Biotechnol. Adv.
Humphrey, A.M., 2004. Chlorophyll as a color and functional 31, 1532e1542.
ingredient. J. Food Sci. Eng. 69, 422e425. Martins, A., Veieira, H., Gaspar, H., Santos, S., 2014. Mar-
Hussein, G., Sankawa, U., Goto, H., Matsumoto, K., keted marine natural products in the pharmaceutical
Watanabe, H., 2006. Astaxanthin, a carotenoid with po- and cosmeceutical industries: tips for success. Mar. Drugs
tential in human health and nutrition. J. Nat. Prod. 69, 12, 1066e1101.
443e449. Mata, T.M., Martins, A.A., Caetano, N.S., 2010. Microalgae
Jaime, L., Rodríguez-Meizoso, I., Cifuentes, A., Santoyo, S., for biodiesel production and other applications: a
Suarez, S., Ib ~ ez, E., Se~
an norans, F.J., 2010. Pressurized liq- review. Renew. Sust. Energ. Rev. 14, 217e232.
uids as an alternative process to antioxidant carotenoids’ Miazek, K., Ledakowicz, S., 2013. Chlorophyll extraction
extraction from Haematococcus pluvialis microalgae. LWT from leaves, needles and microalgae: a kinetic approach.
Food Sci. Technol. 43, 105e112. Int. J. Agric. Biol. Eng. 6, 107e115.
Jeffrey, S.W., Wright, S.W., 2005. Photosynthetic pigments in Miyashita, K., 2009. Function of marine carotenoids. Forum
marine microalgae: insights from cultures and the sea. In: Nutr. 61, 136e146.
Subba Rao, D.V. (Ed.), Algal Cultures Analogues of Moran, N.A., Jarvik, T., 2010. Lateral transfer of genes from
Blooms and Applications. Science Publishers, New fungi underlies carotenoids production in aphids. Science
Hampshire, pp. 33e90. 328, 624e627.
 REFERENCES 243
Mulders, K.J.M., Lamers, P.P., Martens, D.E., Wijffels, R.H., Simopoulos, A.P., 2010. Nutrigenetics/nutrigenomics. Annu.
2014. Phototropic pigment production with microalgae: Rev. Public Health 31, 53e68.
biological constraints and opportunities. J. Phycol. 50, Skjanes, K., Rebours, C., Lindblad, P., 2013. Potential for
229e242. green microalgae to produce hydrogen, pharmaceuticals
Natrah, F., Yosoff, F.M., Shariff, M., Abas, F., Mariana, N.S., and other high value products in a combined process.
2007. Screening of Malaysian indigenous microalgae for Crit. Rev. Biotechnol. 33, 172e215.
antioxidant properties and nutritional value. J. Appl. Spolaore, P., Joannis-Cassan, C., Duran, E., Isambert, A.,
Phycol. 19, 711e718. 2006. Commercial applications of microalgae. J. Biosci.
Norsker, N.H., Barbosa, M.J., Vermue, M.H., Wijffels, R.H., Bioeng. 101, 87e96.
2011. Microalgal productionda close look at the Stahl, W., Sies, H., 2003. Antioxidant activity of carotenoids.
economics. Biotechnol. Adv. 29, 24e27. Mol. Aspect. Med. 24, 345e351.
Pangestuti, R., Kim, S.K., 2011. Biological activities and Stengel, D.B., Connan, S., Popper, Z.A., 2011. Algal chemodi-
health benefit effects of natural pigments derived from versity and bioactivity: sources of natural variability and
marine algae. J. Funct. Foods 3, 255e266. implications for commercial application. Biotechnol. Adv.
Plaza, M., Herrero, M., Cifuentes, A., Ibanez, E., 2009. Inno- 29, 483e501.
vative natural functional ingredients from microalgae. Takaichi, S., 2011. Carotenoids in algae: distributions, biosyn-
J. Agric. Food Chem. 57, 7159e7170. thesis and functions. Mar. Drugs 9, 1101e1118.
Polyakov, N.E., Leshina, T.V., Kovalova, T.A., Kispert, L.D., Takashi, M., 2011. Carotenoids in marine animals. Mar.
2001. Carotenoids as scavengers of free radicals in a Fen- Drugs 9, 278e293.
ton reaction: antioxidants or pro-oxidants? Free Rad. Biol. Vanthoor-Koopmans, M., Wijffels, R.H., 2013. Biorefinery of
Med. 31, 398e404. microalgae for food and fuel. Biores. Technol. 135,
Rasala, B.A., Chao, S.S., Pier, M., Barrera, D.J., Mayfield, S.P., 142e149.
2014. Enhanced genetic tools for engineering multigene Vilchez, C., Forjan, E., Cuaresma, M., Bedmar, F., Garbayo, I.,
traits into green algae. PLoS One 9, e94028. Vega, J.M., 2011. Marine carotenoids: biological functions
Reyes, F.A., Mendiola, J.A., Ibanez, E., del Valle, J.M., 2014. and commercial applications. Mar. Drugs 9, 319e333.
Astaxanthin extraction from Haematococcus pluvialis using Watanabe, M., Ikeuchi, M., 2013. Phycobilisome: achitecture
CO2-expanded ethanol. J. Supercrit. Fluid 92, 75e83. of a light-harvesting supercomplex. Phytother. Res. 116,
Riccioni, G., D’Orazio, N., Franceschelli, S., Speranza, L., 265e276.
2011. Marine carotenoids and cardiovascular risk Wijffels, R.H., Kruse, O., Klaas, J.H., 2013. Potential of indus-
markers. Mar. Drugs 9, 1166e1175. trial biotechnology with cyanobacteria and eukaryotic
Richmond, A., 2004. Handbook of Microalgal Culture: microalgae. Curr. Opin. Biotechnol. 24, 405e413.
Biotechnology and Applied Phycology. Blackwell Science Wright, S.W., Jeffrey, S.W., 2006. Pigment markers for phyto-
Ltd, IA. plankton production. In: Volkman, J.K. (Ed.), Marine
Rodriguez-Sanchez, R., Ortiz-Butron, R., Blas-Valdivia, V., Organic Matter. Springer Verlag, Berlin, Heidelberg,
Hernandez-Garcia, A., Cano-Europa, E., 2012. Phycobili- pp. 71e104.
proteins or C-phycocyanin of Arthrospira (Spirulina) max- Wu, Z., Wu, S., Shi, X., 2007. Supercritical fluid extraction
ima protect against HgCl2-caused oxidative stress and and determination of lutein in heterotrophically culti-
renal damage. Food Chem. 135, 2359e2365. vated Chlorella pyrenoidosa. J. Food Proc. Eng. 30,
Roy, K.R., Arunasree, K.M., Dhoot, A., Aparna, R., 174e185.
Reddy, G.V., Vali, S., Reddanna, P., 2007. C-phycocyanin Yaakob, Z., Ali, E., Zainal, A., Mohamad, M., Takriff, M.S.,
inhibits 2-acetylaminofluorene-induced expression of 2014. An overview: biomolecules from microalgae
MDR1 in mouse macrophage cells: ROS mediated for animal feed and aquaculture. J. Biolog. Res.
pathway determined via combination of experimental Thessaloniki 21, 6.
and in silico analysis. Arch. Biochem. Bioph. 459, Zittelli, G.C., Biondi, N., Rodolfi, L., Tredici, M.R., 2013.
169e177. Photobioreactors for mass production of microalgae.
Roy, S., Llewellyn, C.A., Egeland, E.S., Johnson, G., 2011. In: Richmond, A., Hu, Q. (Eds.), Handbook of
Phytoplankton Pigments: Characterization, Chemotax- Microalgae Culture: Applied Phycology and Biotech-
onomy, and Applications in Oceanography. Cambridge nology, Second ed. Wiley Blackwell, Chichester,
University Press, New York, NY. pp. 225e266.